Recent reports that HSCs can regenerate infarcted myocardium have unleashed a tidal wave of enthusiasm for translating these findings to the clinical arena. A number of institutions have already initiated studies of cytokine or HSC therapy in patients with acute MI. It is our opinion that this rush to clinical trials is not only premature but possibly counterproductive, and that further, careful preclinical investigation is necessary to establish the efficacy of various treatment protocols and the underlying mechanisms. For example, nothing is currently known regarding whether i.v. HSC administration is effective, which cytokine or combination of cytokines is more likely to succeed, how long the window of efficacy is, and whether HSCs are effective in the context of reperfusion. Similarly, nothing is known regarding the nature of the integrin/adhesion molecule interactions that underlie the homing of HSCs or the potential synergistic role of accessory calls, specifically, the FCs. Our fundamental hypothesis is that both i.v. injected and cytokine-mobilized HSCs can regenerate infarcted myocardium and that this process is mediated by a well-defined cascade of molecular interactions that involve specific adhesion molecules (ICAM-1, VCAM-1, P-selectin, and E-selectin), integrins (VLA-4, VLA-5, and LFA-1), chemokines (SDF-1), and chemokine receptors (CXCR-4). We further propose that FCs exert an important potentiating effect on HSC-dependent cardiac reqeneration and that this process can be further enhanced by FL These hypotheses will be tested in a well-established murine model using a broad multidisciplinary approach that will encompass diverse techniques (integrative physiology, protein chemistry, biochemistry, pathology, confocal microscopy, immunohistochemistry, molecular immunology, molecular biology, and gene therapy). Aim I will assess the effectiveness of two clinically-relevant forms of HSC therapy (i.v. HSCs and cytokines) in two different models designed to simulate reperfused and nonreperfused MI and will define the time-window of efficacy. Three clinically-applicable cytokines (G-CSF, SCF, FL) will be tested, alone or in combination. Using gene targeted mice and immunologic blockade of integrins, Aim 2 will systematically investigate the role of four specific adhesion molecules (VCAM-1, ICAM-1, P-selectin, and E-selectin) and three specific integrins (VLA-4, VLA-5, and LFA-1) in HSC migration to the infarcted myocardium. Aim 3 will decipher the role of SDF-1/CXCR-4 interactions in HSC homing and infarct repair, using adenovirus-mediated gene transfer of SDF-1 and CXCR-4. Aim 4 will explore the differential regenerative capacity of HSCs and FCs harvested from bone marrow vis-a-vis peripheral blood and the underlying mechanisms. Aim 5 will determine whether FCs induce allogeneic graft tolerance to HSCs via a Th2 cytokine (IL-4 and IL-10)-dependent mechanism. This proposal will yield novel information regarding the ability of i.v. HSCs and various cytokine regimens to regenerate infarcted myocardium, the molecular mechanisms for HSC homing, the potential role of FCs, and the beneficial effects of FL. The results may eventually lead to the development of novel therapeutic strategies in patients with ischemic heart disease.